Extracellular ATP Signaling Is Mediated by H2O2 and Cytosolic Ca2+ in the Salt Response of Populus euphratica Cells

Extracellular ATP (eATP) has been implicated in mediating plant growth and antioxidant defense; however, it is largely unknown whether eATP might mediate salinity tolerance. We used confocal microscopy, a non-invasive vibrating ion-selective microelectrode, and quantitative real time PCR analysis to evaluate the physiological significance of eATP in the salt resistance of cell cultures derived from a salt-tolerant woody species, Populus euphratica. Application of NaCl (200 mM) shock induced a transient elevation in [eATP]. We investigated the effects of eATP by blocking P2 receptors with suramin and PPADS and applying an ATP trap system of hexokinase-glucose. We found that eATP regulated a wide range of cellular processes required for salt adaptation, including vacuolar Na⁺ compartmentation, Na⁺/H⁺ exchange across the plasma membrane (PM), K⁺ homeostasis, reactive oxygen species regulation, and salt-responsive expression of genes related to K⁺/Na⁺ homeostasis and PM repair. Furthermore, we found that the eATP signaling was mediated by H₂O₂ and cytosolic Ca²⁺ released in response to high salt in P. euphratica cells. We concluded that salt-induced eATP was sensed by purinoceptors in the PM, and this led to the induction of downstream signals, like H₂O₂ and cytosolic Ca²⁺, which are required for the up-regulation of genes linked to K⁺/Na⁺ homeostasis and PM repair. Consequently, the viability of P. euphratica cells was maintained during a prolonged period of salt stress.     

Overexpression of WsSGTL1 Gene of Withania somnifera Enhances Salt Tolerance,Heat Tolerance and Cold Acclimation Ability in Transgenic Arabidopsis Plants

Background

Sterol glycosyltrnasferases (SGT) are enzymes that glycosylate sterols which play important role in plant adaptation to stress and are medicinally important in plants like Withania somnifera. The present study aims to find the role of WsSGTL1 which is a sterol glycosyltransferase from W. somnifera, in plant’s adaptation to abiotic stress.

Methodology

The WsSGTL1 gene was transformed in Arabidopsis thaliana through Agrobacterium mediated transformation, using the binary vector pBI121, by floral dip method. The phenotypic and physiological parameters like germination, root length, shoot weight, relative electrolyte conductivity, MDA content, SOD levels, relative electrolyte leakage and chlorophyll measurements were compared between transgenic and wild type Arabidopsis plants under different abiotic stresses - salt, heat and cold. Biochemical analysis was done by HPLC-TLC and radiolabelled enzyme assay. The promoter of the WsSGTL1 gene was cloned by using Genome Walker kit (Clontech, USA) and the 3D structures were predicted by using Discovery Studio Ver. 2.5.

Results

The WsSGTL1 transgenic plants were confirmed to be single copy by Southern and homozygous by segregation analysis. As compared to WT, the transgenic plants showed better germination, salt tolerance, heat and cold tolerance. The level of the transgene WsSGTL1 was elevated in heat, cold and salt stress along with other marker genes such as HSP70, HSP90, RD29, SOS3 and LEA4-5. Biochemical analysis showed the formation of sterol glycosides and increase in enzyme activity. When the promoter of WsSGTL1 gene was cloned from W. somnifera and sequenced, it contained stress responsive elements. Bioinformatics analysis of the 3D structure of the WsSGTL1 protein showed functional similarity with sterol glycosyltransferase AtSGT of A. thaliana.

Conclusions

Transformation of WsSGTL1 gene in A. thaliana conferred abiotic stress tolerance. The promoter of the gene in W.somnifera was found to have stress responsive elements. The 3D structure showed functional similarity with sterol glycosyltransferases.     

高表达拟南芥miR396提高烟草抗旱性

MiR396是一个由21个核苷酸组成的单链非编码RNA小分子。烟草内的miR396受干旱诱导说明其可能参与烟草的干旱应答。在35S强启动子作用下我们将miR396转入到烟草体内获得高表达转基因植株,生理学测试表明高表达miR396的转基因烟草耐旱性增强,同时叶片表现出比野生型较低的失水率和较高的相对含水量,进一步分析表明转基因植株除了叶片变得更为窄小外,其气孔密度和气孔系数都比野生型降低,这些都表明miR396作为一个正调节因子参与烟草的干旱胁迫应答。     

胡杨谷胱甘肽过氧化物酶PeGPX基因的克隆及转化植株耐盐性分析

胡杨(Populus euphratica Oliv.)具有极强抗盐碱能力。本实验室前期胡杨微阵列芯片数据结果显示:盐胁迫下,胡杨谷胱甘肽过氧化物酶基因(PeGPX)的转录上调,暗示该基因可能对胡杨耐盐性具有一定的作用。为分析 GPX 对植物耐盐性的贡献,本研究以胡杨为材料,利用 RT-PCR 方法克隆了胡杨谷胱甘肽过氧化物酶PeGPX基因,并在烟草中过量表达该基因,以分析谷胱甘肽过氧化物酶活性与植物耐盐性的关系。研究结果显示,实验中克隆的 cDNA (PeGPX)编码谷胱甘肽过氧化物酶,其 ORF 为 693 bp,其蛋白由 231 个氨基酸编码。过量表达 PeGPX 基因的烟草与野生型烟草的耐盐性实验结果显示,野生型烟草植株在加 NaCl(200 mmol/L)的 MS 培养基中生长 15 d 后,无明显的长高,且不长根;而转基因烟草在同样的加盐培养基上,生长基本没有受到抑制,植株生长状态良好,并且能够长根。光合数据显示,在盐胁迫下过量表达 PeGPX 基因烟草的净光合速率受到影响明显小于野生型烟草的净光合速率。酶活数据显示,转基因株系 GPX 酶活与野生型的相比在盐胁迫下活性有非常显著的提高。我们的研究结果说明:过表达 PeGPX 基因使得烟草的耐盐性得到显著提高,这对深入研究PeGPX基因在胡杨耐盐机制中的作用具有重要的意义。高,这对深入研究PeGPX基因在胡杨耐盐机制中的作用具有重要的意义。     

过量表达棉花CBF2基因提高转基因拟南芥抗旱耐盐能力

CBF/DREB是一类植物中特有的转录因子,在植物抵抗逆境胁迫过程中发挥重要功能。本研究从陆地棉(Gossypium hirsutum L.)Coker 312中克隆获得1个棉花CBF/DREB基因,命名为Gh CBF2,该基因编码一个由216个氨基酸组成的CBF蛋白。序列分析结果显示,Gh CBF2与其他植物的CBF蛋白类似,含有AP2转录因子典型的保守结构域。干旱或高盐胁迫处理明显增加了Gh CBF2基因的表达量。亚细胞定位分析结果发现Gh CBF2定位在细胞核中。将Gh CBF2基因构建到由35S启动子调控的植物表达载体p MD上并转化拟南芥(Arabidopsis thaliana L.),结果表明,在干旱和盐胁迫条件下,过量表达Gh CBF2基因拟南芥的成活率显著高于野生型,并且游离脯氨酸和可溶性糖含量也高于野生型,说明转Gh CBF2基因提高了拟南芥的耐盐抗旱能力。采用实时荧光定量PCR方法分析胁迫相关标记基因COR15A、RD29A和ERD6的表达情况,结果显示转基因株系中的表达量显著高于野生型,说明Gh CBF2参与调控拟南芥干旱和盐胁迫相关基因的表达。     

Heterologous Expression of Wheat VERNALIZATION 2 (TaVRN2) Gene in Arabidopsis Delays Flowering and Enhances Freezing Tolerance

The vernalization gene 2 (VRN2), is a major flowering repressor in temperate cereals that is regulated by low temperature and photoperiod. Here we show that the gene from Triticum aestivum (TaVRN2) is also regulated by salt, heat shock, dehydration, wounding and abscissic acid. Promoter analysis indicates that TaVRN2 regulatory region possesses all the specific responsive elements to these stresses. This suggests pleiotropic effects of TaVRN2 in wheat development and adaptability to the environment. To test if TaVRN2 can act as a flowering repressor in species different from the temperate cereals, the gene was ectopically expressed in the model plant Arabidopsis. Transgenic plants showed no alteration in morphology, but their flowering time was significantly delayed compared to controls plants, indicating that TaVRN2, although having no ortholog in Brassicaceae, can act as a flowering repressor in these species. To identify the possible mechanism by which TaVRN2 gene delays flowering in Arabidopsis, the expression level of several genes involved in flowering time regulation was determined. The analysis indicates that the late flowering of the 35S::TaVRN2 plants was associated with a complex pattern of expression of the major flowering control genes, FCA, FLC, FT, FVE and SOC1. This suggests that heterologous expression of TaVRN2 in Arabidopsis can delay flowering by modulating several floral inductive pathways. Furthermore, transgenic plants showed higher freezing tolerance, likely due to the accumulation of CBF2, CBF3 and the COR genes. Overall, our data suggests that TaVRN2 gene could modulate a common regulator of the two interacting pathways that regulate flowering time and the induction of cold tolerance. The results also demonstrate that TaVRN2 could be used to manipulate flowering time and improve cold tolerance in other species.     

低温诱导蛋白及其与植物的耐寒性研究进展

低温诱导蛋白是植物在温度逆境条件下诱导产生的一系列蛋白,以抗冻蛋白、脱水蛋白、热激蛋白和热稳定蛋白较多,而且低温诱导蛋白质一旦在体内形成,植物体就会尽快地适应外界环境,表现出较强的抗逆性.本文对几种主要的低温诱导蛋白——抗冻蛋白、脱水蛋白、热激蛋白和热稳定蛋白的特性及其与植物耐寒性的关系研究进行综述,以期为进一步阐明植物耐寒的分子机制以及提高植物耐寒力研究提供新的思路.     

STCH4/REIL2 Confers Cold Stress Tolerance in Arabidopsis by Promoting rRNA Processing and CBF Protein Translation

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Modifications of Sphingolipid Content Affect Tolerance to Hemibiotrophic and Necrotrophic Pathogens by Modulating Plant Defense Responses in Arabidopsis

Sphingolipids are emerging as second messengers in programmed cell death and plant defense mechanisms. However, their role in plant defense is far from being understood, especially against necrotrophic pathogens. Sphingolipidomics and plant defense responses during pathogenic infection were evaluated in the mutant of long-chain base phosphate (LCB-P) lyase, encoded by the dihydrosphingosine-1-phosphate lyase1 (AtDPL1) gene and regulating long-chain base/LCB-P homeostasis. Atdpl1 mutants exhibit tolerance to the necrotrophic fungus Botrytis cinerea but susceptibility to the hemibiotrophic bacterium Pseudomonas syringae pv tomato (Pst). Here, a direct comparison of sphingolipid profiles in Arabidopsis (Arabidopsis thaliana) during infection with pathogens differing in lifestyles is described. In contrast to long-chain bases (dihydrosphingosine [d18:0] and 4,8-sphingadienine [d18:2]), hydroxyceramide and LCB-P (phytosphingosine-1-phosphate [t18:0-P] and 4-hydroxy-8-sphingenine-1-phosphate [t18:1-P]) levels are higher in Atdpl1-1 than in wild-type plants in response to B. cinerea. Following Pst infection, t18:0-P accumulates more strongly in Atdpl1-1 than in wild-type plants. Moreover, d18:0 and t18:0-P appear as key players in Pst- and B. cinerea-induced cell death and reactive oxygen species accumulation. Salicylic acid levels are similar in both types of plants, independent of the pathogen. In addition, salicylic acid-dependent gene expression is similar in both types of B. cinerea-infected plants but is repressed in Atdpl1-1 after treatment with Pst. Infection with both pathogens triggers higher jasmonic acid, jasmonoyl-isoleucine accumulation, and jasmonic acid-dependent gene expression in Atdpl1-1 mutants. Our results demonstrate that sphingolipids play an important role in plant defense, especially toward necrotrophic pathogens, and highlight a novel connection between the jasmonate signaling pathway, cell death, and sphingolipids.Plants have evolved a complex array of defenses when attacked by microbial pathogens. The success of plant resistance first relies on the capacity of the plant to recognize its invader. Among early events, a transient production of reactive oxygen species (ROS), known as the oxidative burst, is characteristic of successful pathogen recognition (Torres, 2010). Perception of pathogen attack then initiates a large array of immune responses, including modification of cell walls, as well as the production of antimicrobial proteins and metabolites like pathogenesis-related (PR) proteins and phytoalexins, respectively (Schwessinger and Ronald, 2012). The plant hormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are key players in the signaling networks involved in plant resistance (Bari and Jones, 2009; Tsuda and Katagiri, 2010; Robert-Seilaniantz et al., 2011). Interactions between these signal molecules allow the plant to activate and/or modulate an appropriate array of defense responses, depending on the pathogen lifestyle, necrotroph or biotroph (Glazebrook, 2005; Koornneef and Pieterse, 2008). Whereas SA is considered essential for resistance to (hemi)biotrophic pathogens, it is assumed that JA and ET signaling pathways are important for resistance to necrotrophic pathogens in Arabidopsis (Arabidopsis thaliana; Thomma et al., 2001; Glazebrook, 2005). A successful innate immune response often includes the so-called hypersensitive response (HR), a form of rapid programmed cell death (PCD) occurring in a limited area at the site of infection. This suicide of infected cells is thought to limit the spread of biotrophic pathogens, including viruses, bacteria, fungi, and oomycetes (Mur et al., 2008).During the past decade, significant progress has been made in our understanding of the cellular function of plant sphingolipids. Besides being structural components of cell membranes, sphingolipids are bioactive metabolites that regulate important cellular processes such as cell survival and PCD, occurring during either plant development or plant defense (Dunn et al., 2004; Berkey et al., 2012; Markham et al., 2013). The first evidence of the role of sphingolipids in these processes came from the use of the fungal toxins fumonisin B1 (FB1) and AAL, produced by the necrotrophic agent Alternaria alternata f. sp. lycopersici. These toxins are structural sphingosine (d18:1) analogs and function as ceramide synthase inhibitors. They triggered PCD when exogenously applied to plants. Mutant strains in which the production of such toxins is abrogated failed to infect the host plant, implying that toxin accumulation is required for pathogenicity and that the induction of plant PCD could be considered a virulence tool used by necrotrophic pathogens (Berkey et al., 2012). Moreover, several studies revealed that ceramides (Cers) and long-chain bases (LCBs) are also potent inducers of PCD in plants. For example, exogenously applied Cers and LCBs (d18:0, d18:1, or t18:0) induced PCD either in cell suspension cultures (Liang et al., 2003; Lachaud et al., 2010, 2011; Alden et al., 2011) or in whole seedlings (Shi et al., 2007; Takahashi et al., 2009; Saucedo-García et al., 2011). AAL- and FB1-induced PCD seemed to be due to the accumulation of free sphingoid bases (dihydrosphingosine [d18:0] and phytosphingosine [t18:0]; Abbas et al., 1994; Brandwagt et al., 2000; Shi et al., 2007). Spontaneous cell death in lag one homolog1 or l-myoinositol1-phosphate synthase mutant could be due to trihydroxy-LCB and/or Cer accumulation (Donahue et al., 2010; Ternes et al., 2011). Deciphering of Cer participation in the induction of HR and associated PCD also came from studies on accelerated cell death5 (acd5) and enhancing resistance to powdery mildew8 (RPW8)-mediated hypersensitive response (erh1) mutants, which displayed overaccumulation of Cers. These mutants exhibited spontaneous cell death and resistance to biotrophic pathogens, which seemed to be linked with SA and PR protein accumulation (Liang et al., 2003; Wang et al., 2008).Altogether, these data provide evidence of a link between PCD, defense, and sphingolipid metabolism. However, the fatty acid hydroxylase1/2 (atfah1/atfah2) double mutant that accumulates SA and Cers was more tolerant to the obligate biotrophic fungus Golovinomyces cichoracearum but did not display a PCD-like phenotype, suggesting that Cers alone are not involved in the induction of PCD (König et al., 2012). Moreover, Saucedo-García et al. (2011) postulated that dihydroxy-LCBs, but not trihydroxy-LCBs, might be primary mediators for LCB-induced PCD. The sphingoid base hydroxylase sbh1/sbh2 double mutant completely lacking trihydroxy-LCBs showed enhanced expression of PCD marker genes (Chen et al., 2008). On the contrary, increase in t18:0 was specifically sustained in plant interaction with the avirulent Pseudomonas syringae pv tomato (Pst) strain and correlated with a strong PCD induction in leaves (Peer et al., 2010). Thus, the nature of sphingolipids able to induce PCD is still under debate and may evolve depending on plants and their environment. The phosphorylated form of LCBs (LCB-Ps) could abrogate PCD induced by LCBs, Cers, or heat stress in a dose-dependent manner (Shi et al., 2007; Alden et al., 2011). Furthermore, blocking the conversion of LCBs to LCB-Ps by using specific inhibitors induced PCD in cell suspension culture (Alden et al., 2011). Recently, overexpression of rice (Oryza sativa) LCB kinase in transgenic tobacco (Nicotiana tabacum) plants reduced PCD after treatment with FB1 (Zhang et al., 2013). Genetic mutation on LCB-P lyase encoded by the AtDPL1 gene, modifying the LCB-LCB-P ratio, could impact PCD levels after treatment with FB1 (Tsegaye et al., 2007). Altogether, these data point to the existence of a rheostat between LCBs and their phosphorylated forms that controls plant cell fate toward cell death or survival.Data on plant sphingolipid functions are still fragmentary. Only a few reports have described interconnections between sphingolipids, cell death, and plant defense responses, almost exclusively in response to (hemi)biotrophic pathogens. Knowledge about such relations in response to necrotrophic pathogens is still in its infancy (Rivas-San Vicente et al., 2013; Bi et al., 2014). In this report, the link between sphingolipids, cell death, and plant defense has been explored in response to Botrytis cinerea infection and in comparison with Pst infection. For this purpose, Atdpl1 mutant plants, disturbed in LCB/LCB-P accumulation without displaying any phenotype under standard growth conditions (Tsegaye et al., 2007), have been analyzed after pathogen infection. Our results revealed that modification of sphingolipid contents not only impacted plant tolerance to hemibiotrophs but also greatly affected resistance to necrotrophs. Whereas the SA signaling pathway is globally repressed in Atdpl1-1 compared with wild-type plants, the JA signaling pathway is significantly enhanced. Cell death and ROS accumulation are markedly modified in Atdpl1-1 mutant plants. We further demonstrated that phytosphingosine-1-phosphate (t18:0-P) and d18:0 are key players in pathogen-induced cell death and ROS generation. Here, we thus established a link between JA signaling, PCD, and sphingolipid metabolism.     

拟南芥转录因子CBF的冷调控机制研究进展

拟南芥中CBF(C-repeat binding factor)转录因子在抗寒性方面起重要作用,低温可诱导CBF转录因子的表达。CBF转录因子能够特异结合启动子中含有CRT/DRE(C-repeat/dehydration responsive element)的顺式元件,激活COR等基因的表达,从而增强植株抗寒能力,对调控逆境诱导基因的表达具有非常重要的作用。对CBF转录因子的结构特点、功能、表达调控以及与CBF相关的其它低温调节途径进行了综述,为提高植物综合抗逆性的研究提供参考。     

The Arabidopsis AtAAE13.1 Gene Enhances Salt Stress Tolerance in Angiosperms and Gymnosperm Plant Cells

In Vitro Cellular & Developmental Biology - Plant - The Arabidopsis malonyl-CoA synthetase gene AAE13.1 (AtAAE13.1) plays important roles in cell metabolism, plant growth and development, and...     

Exogenous Abscisic Acid Alleviates Cadmium Toxicity by Restricting Cd2+ Influx in Populus euphratica Cells

Abscisic acid (ABA), a widely known phytohormone involved in the plant response to abiotic stress, plays a vital role in mitigating Cd²⁺ toxicity in herbaceous species. However, the role of ABA in ameliorating Cd²⁺ toxicity in woody species is largely unknown. In the present study, we investigated ABA restriction on Cd²⁺ uptake and the relevance to Cd²⁺ stress alleviation in Cd²⁺-hypersensitive Populus euphratica. ABA (5 μM) markedly improved cell viability and growth but reduced membrane permeability in CdCl₂ (100 μM)-stressed P. euphratica cells. Moreover, ABA significantly increased the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX), contributing to the scavenging of Cd²⁺-elicited H₂O₂ within P. euphratica cells during the period of CdCl₂ exposure (100 μM, 24–72 h). ABA alleviation of Cd²⁺ toxicity was mainly the result of ABA restriction of Cd²⁺ uptake under Cd²⁺ stress. Steady-state and transient flux recordings showed that ABA inhibited Cd²⁺ entry into Cd²⁺-shocked (100 μM, 30 min) and short-term-stressed P. euphratica cells (100 μM, 24–72 h). Non-invasive micro-test technique data showed that H₂O₂ (3 mM) stimulated the Cd²⁺-elicited Cd²⁺ influx but that the plasma membrane (PM) Ca²⁺ channel inhibitor LaCl₃ blocked it, suggesting that the Cd²⁺ influx was through PM Ca²⁺-permeable channels. These results suggested that ABA up-regulated antioxidant enzyme activity in Cd²⁺-stressed P. euphratica and that these enzymes scavenged the Cd²⁺-elicited H₂O₂ within cells. The entry of Cd²⁺ through the H₂O₂-mediated Ca²⁺-permeable channels was subsequently restricted; thus, Cd²⁺ buildup and toxicity were reduced in the Cd²⁺-hypersensitive species, P. euphratica.

     

Role of Tonoplast Proton Pumps and Na+/H+ Antiport System in Salt Tolerance of Populus euphratica Oliv.

Populus euphratica has been used as a plant model to study resistance against salt and osmotic stresses, with recent studies having characterized the tonoplast and the plasma membrane ATPases, and two Na⁺/H⁺ antiporters, homologs of the Arabidopsis tonoplast AtNHX1, were published in databases. In the present work we show that P. euphratica suspension-cultured cells are highly tolerant to high salinity, being able to grow with up to 150 mM NaCl in the culture medium without substantial modification of the final population size when compared to the control cells in the absence of salt. At a salt concentration of 300 mM, cells were unable to grow but remained highly viable up to 17 days after subculture. The addition of a 1-M-NaCl pulse to unadapted cells did not promote a significant loss in cell viability within 48 h. In tonoplast vesicles purified from cells cultivated in the absence of salt and from salt-stressed cells, vacuolar H⁺-pyrophosphatase (V-H⁺-PPase) seemed to be the primary tonoplast proton pump; however, there appears to be a decrease in V-H⁺-PPase activity with exposure to NaCl, in contrast to the sodium-induced increase in the activity of vacuolar H⁺-ATPase (V-H⁺-ATPase). Despite reports that in P. euphratica there is no significant difference in the concentration of Na⁺ in the different cell compartments under NaCl stress, in the present study, confocal and epifluorescence microscopic observations using a Na⁺-sensitive probe showed that suspension-cultured cells subject to a salt pulse accumulated Na⁺ in the vacuole when compared with control cells. Concordantly, a tonoplast Na⁺/H⁺ exchange system is described whose activity is upregulated by salt and, indirectly, by a salt-mediated increase of V-H⁺-ATPase activity.